The long term objective of this research is to define the rules that govern the interaction of small molecules with DNA in chromatin. An understanding of how chromatin structures affect the biochemistry of genotoxic agents is essential to the design of selective chemotherapeutic agents and probes of chromatin structure, and to the understanding of the mechanisms of chemical mutagenicity and carcinogenicity. The proposed experiments will address the role of nucleosome structure on the DNA damage produced by two radical-mediated DNA cleaving agents, neocarzinostatin (NCS) and bleomycin (BLM), in a model chromatin system, the rDNA of Tetrahymena thermophila. Chromatin structures in the central spacer of the rDNA will be mapped by enzymatic footprinting techniques, and the sites of DNA damage produced by NCS and BLM compared to the chromatin structures. The effects of nucleosome structure on the sequence-specificity, chemistry, and single- and double-strand nature of the DNA damage will be assessed by genomic sequencing. The physical determinants of cleavage location will be studied by comparing NCS- and BLM-mediated DNA damage to that produced by the related enediynes, calicheamicin and esperamicin, and the intercalating agent, ethidium bromide. Binding of the latter agent will be assessed by photochemical cleavage. Finally, the binding of NCS to nucleosome cores and chromatosomes will be studied by two fluorescence quenching techniques.